The inherited bleeding disorder hemophilia A results from a deficiency of plasma clotting factor VIII. Affected patients experience significant morbidity due to repeated joint hemorrhages and subsequent arthropathy and there is increased mortality related to life-threatening bleeding events. The majority of patients are treated with episodic or prophylactic infusions of recombinant FVIII (rFVIII). The ability to bioengineer recombinant clotting factors with improved function holds promise to overcome some of the limitations in current treatment, the high costs of therapy and increase availability to a broader world hemophilia population. These novel molecules partnered with advances in gene transfer vector design and delivery may ultimately achieve persistent expression of FVIII leading to an effective long-term treatment strategy for hemophilia A. In addition, these novel FVIII molecules could be partnered with new advances in alternative recombinant protein production in transgenic animals yielding an affordable, more abundant supply of rFVIII. The long term goal of the proposed research is to advance several novel bioengineered forms of rFVIII developed in our laboratory to readiness for a clinical trial in hemophilia A. Our research in the past has focused on the limitations of rFVIII synthesis, secretion and regulation of activity. This has given us insights to successfully make targeted modifications to rFVIII to overcome these limitations. The major goals of this proposal are to characterize the in vitro and in vivo properties or rFVIII bioengineered for improved secretion efficiency (Specific Aim 1) and rFVIII bioengineered to be resistant to inactivation (Specific Aim 2). The proposed experiments will use established biochemical assays to characterize the unique properties of these novel molecules as well as murine and canine hemophilia A animal models to characterize their control of bleeding. These novel rFVIII molecules will be produced by standard mammalian cell culture technology as well as in transgenic animals. We will also test whether the bioengineered modifications will increase the risk for antibody formation and adverse clotting events. Findings from the proposed experiments will provide insights into the FVIII secretion pathway, FVIII structure and function and intermolecular interactions in vivo. The results will directly impact on the future of rFVIII therapeutics for hemophilia.